Crystals of azabicyclic compound

ABSTRACT

The crystals are Form II crystals of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide showing an X-ray powder diffraction spectrum having at least three characteristic diffraction peaks at angles (2θ±0.2°) selected from the group consisting of 7.7°, 8.0°, 11.1°, 12.5°, 12.9°, 15.2°, 15.8°, 17.2°, 19.0°, 22.5°, 26.1°, and 27.4°.

FIELD OF THE INVENTION

The present invention relates to a novel crystal of an azabicyclic compound which is stable and has excellent oral absorbability, and is useful as an antitumor agent.

BACKGROUND OF THE INVENTION

In general, when a compound is used as an effective active ingredient of a pharmaceutical, the chemical and physical stability of the compound is required in order to keep the quality stable and/or facilitate the storage management. Therefore, the obtained compound is preferably in a stable crystal form and in general the most stable crystal form is often selected as a drug substance for a pharmaceutical.

So far, a plurality of HSP90 inhibitors have been reported as an antitumor agent, for example, Patent Document 1 and 2 describe 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide (hereinafter also referred to as “Compound 1”) as a compound which has excellent HSP90 inhibitory action and shows antitumor activity.

Meanwhile, although a pharmaceutical composition for oral administration requires in general not only the stability of an effective ingredient, but also excellent absorbability on oral administration, Patent Document 1 and 2 do not describe any crystals of Compound 1, nor the stability and oral absorbability of the crystals.

CITATION LIST Patent Document

-   [Patent Document 1]

International Publication No. WO2012/093708

-   [Patent Document 2]

International Publication No. WO2011/004610

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

An object of the present invention is to provide a crystal of Compound 1 useful as an antitumor agent which is stable and has excellent oral absorbability.

Means for Solving the Problem

To achieve the object, the present inventor synthesized Compound 1 according to the method of manufacturing described in Patent Document 1 to obtain a Form I crystal of Compound 1. However, as described in Examples below, the Form I crystal had problems in oral absorbability, and thus they further investigated the crystallization conditions. As a result, they found that a Form II crystal can be obtained by adding and suspending Compound 1 into a certain organic solvent, and further found that the Form II crystal is superior in stability and oral absorbability compared to the Form I crystal, thereby completing the present invention.

Thus, the present invention relates to provision of the following [1] to [15].

-   [1] A Form II crystal of     3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide     showing an X-ray powder diffraction spectrum having at least three     characteristic diffraction peaks at angles (2θ±0.2°) selected from     the group consisting of 7.7°, 8.0°, 11.1°, 12.5°, 12.9°, 15.2°,     15.8°, 17.2°, 19.0°, 22.5°, 26.1°, and 27.4°. -   [2] The Form II crystal according to [1], wherein the crystal shows     an X-ray powder diffraction spectrum having at least five     characteristic diffraction peaks at angles (2θ±0.2°) selected from     the group consisting of 7.7°, 8.0°, 11.1°, 12.5°, 12.9°, 15.2°,     15.8°, 17.2°, 19.0°, 22.5°, 26.1°, and 27.4°. -   [3] The Form II crystal according to [1] or [2], wherein the     endothermic peak determined by simultaneous thermogravimetric     differential thermal analyzing is around 270° C. -   [4] A pharmaceutical composition comprising the Form II crystal     according to any of [1] to [3]. -   [5] A pharmaceutical composition for oral administration comprising     the Form II crystal according to any of [1] to [3]. -   [6] An antitumor agent comprising the Form II crystal according to     any of [1] to [3]. -   [7] Use of the Form II crystal according to any of [1] to [3], for     the production of a pharmaceutical composition. -   [8] The use according to [7], wherein the pharmaceutical composition     is a pharmaceutical composition for oral administration. -   [9] Use of the Form II crystal according to any of [1] to [3], for     the production of an antitumor agent. -   [10] The Form II crystal according to any of [1] to [3], for use as     a medicament. -   [11] The Form II crystal according to any of [1] to [3], for use in     the treatment of a tumor. -   [12] A method of treating a tumor, comprising administering an     effective amount of the Form II crystal according to any of [1] to     [3] to a subject in need thereof. -   [13] A method of manufacturing the Form II crystal according to any     of [1] to [3], comprising the steps of: -   (1) suspending the     3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide     in an organic solvent with heating to obtain a suspension, and -   (2) obtaining solid-state     3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide     from the suspension obtained in the above step (1). -   [14] The method of manufacturing the Form II crystal according to     [13], wherein the organic solvent is 2-propanol, methyl acetate,     ethyl acetate, propyl acetate, butyl acetate, cyclopentyl methyl     ether, methyl ethyl ketone, methyl isobutyl ketone, acetone,     acetonitrile or a mixed solvent thereof. -   [15] A method of manufacturing the Form II crystal according to any     of [1] to [3], comprising the steps of: -   (1) suspending the     3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide     in an organic solvent selected from the group consisting of     2-propanol, methyl acetate, ethyl acetate, propyl acetate, butyl     acetate, cyclopentyl methyl ether, methyl ethyl ketone, methyl     isobutyl ketone, acetone, acetonitrile and a mixed solvent thereof     to obtain a suspension, and -   (2) obtaining solid-state     3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide     from the suspension obtained in the above (1).

Effect of the Invention

According to the present invention, the Form II crystal of Compound 1 has high stability and excellent oral absorbability, and is useful as an oral pharmaceutical.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 An X-ray powder diffraction spectrum of the Form I crystal of Compound 1 (the vertical axis represents intensity (cps) and the horizontal axis represents the diffraction angle (2θ±0.2°)).

FIG. 2 An X-ray powder diffraction spectrum of the Form II crystal of Compound 1 (the vertical axis represents intensity (cps) and the horizontal axis represents the diffraction angle(2θ±0.2°)).

FIG. 3 A differential scanning calorimetry (DSC) curve of the Form II crystal of Compound 1.

FIG. 4 A result of blood concentration measurement test of the Form II crystal of Compound 1.

DETAILED DESCRIPTION OF THE INVENTION

Compound 1 of the present invention is 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide (represented by the below formula). Compound 1 is known to show HSP90 inhibitory activity and show excellent antitumor activity. Compound 1 can be synthesized based on the production method described in Patent Documents 1 and 2.

The crystal of the present invention may be a crystal comprising the Form II crystal of Compound 1, and may be a single crystal of the Form II crystal or a polymorphic mixture comprising crystals other than the Form II crystal. For the crystal of the present invention, the Form II crystal with high purity is preferred. Specifically, the chemical purity of the Form II crystal is preferably 90% or more, more preferably 95% or more, and particularly preferably 98% or more.

The Form II crystal of the present invention can be obtained by adding and suspending Compound 1 into a certain organic solvent. Specifically, the Form II crystal can be obtained by the production method comprising the following steps (1) and (2):

-   (1) suspending Compound 1 in an organic solvent to obtain a     suspension, -   (2) obtaining solid-state Compound 1 from the suspension obtained in     the above (1).

Although the Compound 1 to be added to an organic solvent may be a crystal or not, crystal form of the Compound 1 is preferably used and in particular the Form II crystal of the Compound 1 is preferably used from the viewpoint of obtaining the Form II crystal with high purity. In crystallization of the present invention, a seed crystal may be used. From the viewpoint of obtaining the Form II crystal with high purity, the Form II crystal is preferred for use as a seed crystal.

Examples of the organic solvent used in the crystallization of the present invention include alcohols such as methanol, n-propanol, 2-propanol and ethylene glycol; aliphatic carboxylate esters such as methyl acetate, ethyl acetate, propyl acetate, and butyl acetate; ethers such as diethyl ether, methyl tert-butyl ether, cyclopentyl methyl ether, 1,4-dioxane, tetrahydrofuran; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, and cyclohexanone; aromatic solvents such as toluene, xylene, and chlorobenzene; aprotic polar organic solvents such as acetonitrile, N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, 1,3-dimethyl-2-imidazolidinone, and dimethylsulfoxide or mixed solvents thereof. Preferably, the organic solvent is a ketone, a monohydric alcohol having 3 or more carbon atoms, a dihydric alcohol, an aliphatic carboxylate ester, an ether, an aprotic polar organic solvent or a mixed solvent thereof, and more preferably is 2-propanol, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, cyclopentyl methyl ether, methyl ethyl ketone, methyl isobutyl ketone, acetone, acetonitrile or a mixed solvent thereof. Particularly preferably, from the viewpoint of purity and yield of the Form II crystal, the organic solvent is methyl acetate, methyl ethyl ketone, acetone or a mixed solvent thereof.

From the viewpoint of purity and yield of the Form II crystal, the amount (v/w) of the organic solvent in the crystallization of the present invention is preferably 2-30 times amount, more preferably 3-20 times amount, and particularly preferably 4-15 times amount, relative to the amount of Compound 1.

Preferably, in the crystallization of the present invention, Compound 1 is added and suspended into an organic solvent with heating and refluxed for a long time. The heating temperature in the crystallization of the present invention is not specifically limited as long as it falls within the temperature suitable for reflux, and is appropriately determined depending on the organic solvent used. Preferably, the heating temperature is 52-126° C.

The duration of reflux in the crystallization of the present invention is preferably 12-60 hours and more preferably 16-48 hours, otherwise, crystallization dose not sufficiently proceed and thus a high purity crystal cannot be obtained in shorter duration, and on the other hand, degradation of the crystal occurs and leads to low yield in longer duration.

In the crystallization of the present invention, precipitate of Form II crystal after cooling can be collected after reflux. Cooling temperature may be appropriately adjusted, is preferably room temperature.

The precipitate of the crystal can be isolated and purified from said dissolution or mixed solution according to known separation and purification methods such as, for example, filtration, washing by an organic solvent, and drying under reduced pressure.

Examples of the organic solvent used for the washing include lower alcohols, acetone, and acetonitrile. The organic solvent used in Form II crystallization may be used for washing.

The Form II crystal of the present invention thus obtained is a crystal showing an X-ray powder diffraction spectrum having 3 or more, preferably 5 or more, more preferably 8 or more, and still more preferably 12 or more characteristic diffraction peaks at angles (2θ±0.2°) selected from the group consisting of 7.7°, 8.0°, 11.1°, 12.5°, 12.9°, 15.2°, 15.8°, 17.2°, 19.0°, 22.5°, 26.1°, and 27.4°, as shown in FIG. 2. The Form II crystal shows an endothermic peak around 270° C., as shown in the result of differential scanning calorimetry (DSC) in FIG. 3.

In contrast, the Form I crystal is a crystal showing an X-ray powder diffraction spectrum having characteristic diffraction peaks at angles (2θ±0.2°) of 8.1°, 12.1°, 14.0°, 16.2°, 21.5°, 25.4° and 28.3°, as shown in FIG. 1.

Peak values in an X-ray powder diffraction spectrum may involve some errors due to measuring instruments or measuring conditions such as reading conditions of peaks. The peak values in the specification of the present application may have measurement errors in the range of around ±0.2°.

The endothermic peak (a peak top value) measured in differential scanning calorimetry may fluctuate depending on the increase of temperature per minute, the amount of samples and purity etc. The term “around” in the specification of the present application refers to ±5.0° C.

As shown in Examples below, the Form I crystal and the Form II crystal of Compound 1 have significantly different absorbability on oral administration. The result that the Form II crystal, a stable form, shows higher oral absorbability compared to the Form I crystal, a metastable form, is an unexpected result because a metastable form in general has higher solubility than a stable form (Akira Tsuji, “New pharmaceutics”, published from NANKODO Co. Ltd.).

The Form I crystal of Compound 1 changes to the Form II crystal by refluxing. The Form II crystal is stable in high temperature conditions and high humidity conditions. Therefore, the Form II crystal is stable compared to the Form I crystal and useful as a pharmaceutical material.

Therefore, the Form II crystal of the present invention is useful as an effective ingredient of a pharmaceutical composition and in particular useful as an effective ingredient of pharmaceutical composition for oral administration. The Form II crystal of the present invention is useful as an antitumor agent, because Compound 1 shows excellent HSP90 inhibitory activity. Target cancers for treatment include, without limitation, head and neck carcinoma, gastrointestinal carcinoma (such as esophageal carcinoma, gastric cancer, gastrointestinal stromal tumor, duodenal carcinoma, hepatocarcinoma, biliary carcinoma (such as gallbladder-bile duct cancer), pancreatic cancer, small intestine carcinoma, and large bowel cancer (such as colorectal carcinoma, colon cancer, and rectal cancer)), lung carcinoma, breast carcinoma, ovarian cancer, uterine carcinoma (such as cervical cancer, and endometrial cancer), kidney carcinoma, bladder cancer, prostate cancer, urothelial carcinoma, bone and soft tissue sarcoma, blood cancer (such as B-cell lymphoma, chronic lymphocytic leukemia, peripheral T-cell lymphoma, myelodysplastic syndrome, acute myeloid leukemia, and acute lymphocytic leukemia), multiple myeloma, skin cancer, and mesothelioma.

When the Form II crystal of the present invention is used as an active ingredient of a pharmaceutical composition, it may be blended with a pharmaceutically acceptable carrier as needed and a variety of administration forms may be employed depending on preventive or therapeutic purposes. Said forms are preferably an oral agent such as a tablet, a capsule, a granule, a subtle granule, and a powdered drug. Each of these administration forms can be manufactured by methods for drug formulation known to those skilled in the art.

EXAMPLES

Although the present invention is specifically described below with reference to Examples, the present invention shall not be limited by these embodiments. Although the present invention is sufficiently described by Examples, it should be understood that various changes and modifications can be made by those skilled in the art. Therefore, such changes and modifications are encompassed by the present invention, as long as they do not depart from the scope of the present invention.

Reagents used in Examples were commercially available, unless otherwise specified. A NMR spectrum was measured by using tetramethylsilane as an internal standard when tetramethylsilane is included in a deuterated solvent and using a NMR solvent as an internal standard otherwise, using AL400 (400 MHz; Japan Electro Optical Laboratory (JEOL)), Mercury 400 (400 MHz; Agilent Technologies) model spectrometer, or Inova 400 (400 MHz; Agilent Technologies) model spectrometer equipped with 400MNMR probe (Protasis). All δ values are indicated in ppm.

The meanings of abbreviation are shown below.

-   s: Singlet -   d: Doublet -   t: Triplet -   q: Quartet -   dd: Double Doublet -   dt: Double Triplet -   td: Triple Doublet -   tt: Triple Triplet -   ddd: Double Double Doublet -   ddt: Double Double Triplet -   dtd: Double Triple Doublet -   tdd: Triple Double Doublet -   m: Multiplet -   br: Broad -   brs: Broad Singlet

X-Ray Powder Diffraction Measurement

X-ray powder diffraction was measured in accordance with the following test conditions after lightly pulverizing a suitable amount of test material by using an agate mortar as needed. Instrument: PANalytical EMPYREAN

-   Target: Cu -   X-ray output setting: 40 mA, 45 kV -   Scanning range: 2.0-40.0° -   Step size: 0.026° -   Divergence slit: automatic -   Irradiation width: 10.00 mm -   Sample width: 10.00 mm

Handling of instruments including data processing was performed in accordance with the method and procedure indicated by each instruments.

Thermal Analysis Measurement (Differential Scanning Calorimetry (DSC))

DSC was performed in accordance with the following test conditions.

-   Instrument: TA Instruments Q1000 -   Sample: around 1 mg -   Sample container: made of aluminum -   Heating rate: heating at 10° C./min to 300° C. -   Atmosphere gas: Nitrogen -   Nitrogen gas flow rate: 50 mL/min.

Handling of instruments including data processing was performed in accordance with the method and procedure indicated by each instruments.

Comparative Example 1 Synthesis of the Form I Crystal of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (3.58 g) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide obtained according to the method of manufacturing described in International Publication No. WO2012/093708 pamphlet and International Publication No. WO2011/004610 pamphlet was added to ethanol (7.84 mL) and stirred for 2 hours at room temperature. After collecting the solid by filtration, the solid was washed with ethanol (7.84 mL), followed by drying under reduced pressure at 70-80° C. for 20 hours to obtain the Form I crystal (yield: 2.40 g, percent yield: 61.2%, purity: 98.21%).

The Form I crystal gave an X-ray powder diffraction spectrum having characteristic peaks at diffraction angles (2θ) of 8.1°, 10.9°, 12.1°, 14.0°, 14.9°, 16.2°, 17.7°, 20.2°, 21.0°, 21.5°, 22.6°, 24.3°, 25.4°, 26.4°, 27.0°, 28.3°, 30.2°, 30.9°, 31.5°, 32.7°, 34.7°, 35.4° and 36.6°, as shown in FIG. 1.

¹H-NMR (DMSO-d₆): δ ppm 9.35 (1H, d, J=4.88 Hz), 8.93 (1H, d, J=1.22 Hz), 8.84 (1H, brs), 8.72 (1H, d, J=1.95 Hz), 8.70 (1H, s), 8.63 (1H, d, J=1.22 Hz), 8.60 (1H, dd, J=8.29, 1.95 Hz), 8.46 (1H, s), 8.25 (1H, d, J=8.29 Hz), 8.22 (1H, brs), 8.12 (1H, d, J=4.88 Hz), 4.59 (3H, s), 3.95 (1H, tt, J=6.83, 6.83 Hz), 3.21 (2H, q, J=7.56 Hz), 1.83(6H, d, J=6.83 Hz), 1.75 (3H, t, J=7.56 Hz): LRMS (ESI) m/z 455 [M+H]

Example 1 Synthesis of the Form II Crystal of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (4.0 g) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide obtained according to the method of manufacturing described in International Publication No. WO2012/093708 pamphlet and International Publication No. WO2011/004610 pamphlet was added to acetone (19.54 mL) and stirred for 16 hours by heating under reflux. After allowing the mixture to cool to room temperature, the solid was collected by filtration and washed with acetone (8.4 mL), followed by drying under reduced pressure at 70-80° C. for 16-24 hours to obtain the Form II crystal (yield: 1.59 g, percent yield: 57.0%, purity 98.37%).

The Form II crystal gave an X-ray powder diffraction spectrum having characteristic peaks at diffraction angles (2θ) of 7.7°, 8.0°, 11.1°, 12.5°, 12.9°, 14.2°, 15.2°, 15.8°, 17.2°, 17.7°, 19.0°, 20.2°, 21.1°, 22.5°, 22.8°, 23.5°, 24.5°, 26.1°, 26.7°, 27.4°, 28.0°, 28.7°, 29.4°, 30.0°, 31.7°, 35.1°, 36.2°, 36.9° and 37.6°, as shown in FIG. 2. The Form II crystal showed an endothermic peak around 270° C., as shown in the result of differential scanning calorimetry (DSC) in FIG. 3.

¹H-NMR (DMSO-d₆): δ ppm 9.35 (1H, d, J=4.88 Hz), 8.93 (1H, d, J=1.22 Hz), 8.84 (1H, brs), 8.72 (1H, d, J=1.95 Hz), 8.70 (1H, s), 8.63 (1H, d, J=1.22 Hz), 8.60 (1H, dd, J=8.29, 1.95 Hz), 8.46 (1H, s), 8.25 (1H, d, J=8.29 Hz), 8.22 (1H, brs), 8.12 (1H, d, J=4.88 Hz), 4.59 (3H, s), 3.95 (1H, tt, J=6.83, 6.83 Hz), 3.21 (2H, q, J=7.56 Hz), 1.83(6H, d, J=6.83 Hz), 1.75 (3H, t, J=7.56 Hz): LRMS (ESI) m/z 455 [M+H]

Example 2 Synthesis of the Form II Crystal of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (400 mg) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide obtained according to the method of manufacturing described in International Publication No. WO2012/093708 pamphlet and International Publication No. WO2011/004610 pamphlet was added to methyl ethyl ketone (2.8 mL) and stirred for 16 hours by heating under reflux. After allowing the mixture to cool to room temperature, the solid was collected by filtration and washed with methyl ethyl ketone (1.2 mL), followed by drying under reduced pressure at 70-80° C. for 16-24 hours to obtain the Form II crystal (yield: 197 mg, percent yield: 60.9%, purity 98.83%).

Example 3 Synthesis of the Form II Crystal of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (400 mg) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide obtained according to the method of manufacturing described in International Publication No. WO2012/093708 pamphlet and International Publication No. WO2011/004610 pamphlet was added to acetonitrile (4.0 mL) and stirred for 3 hours by heating under reflux. After allowing the mixture to cool to room temperature, the solid was collected by filtration and washed with acetonitrile (1.2 mL), followed by drying under reduced pressure at 70-80° C. for 3 hours to obtain the Form II crystal (yield: 120 mg, percent yield: 43.0%, purity 98.25%).

Example 4 Synthesis of the Form II Crystal of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (400 mg) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide obtained according to the method of manufacturing described in International Publication No. WO2012/093708 pamphlet and International Publication No. WO2011/004610 pamphlet was added to methyl isobutyl ketone (4.0 mL) and stirred for 3 hours by heating under reflux. After allowing the mixture to cool to room temperature, the solid was collected by filtration and washed with methyl isobutyl ketone (1.2 mL), followed by drying under reduced pressure at 70-80° C. for 3 hours to obtain the Form II crystal (yield: 154 mg, percent yield: 55.3%, purity 96.89%).

Example 5 Synthesis of the Form II Crystal of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (400 mg) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide obtained according to the method of manufacturing described in International Publication No. WO2012/093708 pamphlet and International Publication No. WO2011/004610 pamphlet was added to 2-propanol (4.0 mL) and stirred for 3 hours by heating under reflux. After allowing the mixture to cool to room temperature, the solid was collected by filtration and washed with 2-propanol (1.2 mL), followed by drying under reduced pressure at 70-80° C. for 3 hours to obtain the Form II crystal (yield: 108 mg, percent yield: 38.8%, purity 96.83%).

Example 6 Synthesis of the Form II Crystal of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (400 mg) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide obtained according to the method of manufacturing described in International Publication No. WO2012/093708 pamphlet and International Publication No. WO2011/004610 pamphlet was added to ethyl acetate (4.0 mL) and stirred for 3 hours by heating under reflux. After allowing the mixture to cool to room temperature, the solid was collected by filtration and washed with ethyl acetate (1.2 mL), followed by drying under reduced pressure at 70-80° C. for 3 hours to obtain the Form II crystal (yield: 156 mg, percent yield: 56.0%, purity 96.45%).

Example 7 Synthesis of the Form II Crystal of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (400 mg) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide obtained according to the method of manufacturing described in International Publication No. WO2012/093708 pamphlet and International Publication No. WO2011/004610 pamphlet was added to butyl acetate (4.0 mL) and stirred for 3 hours by heating under reflux. After allowing the mixture to cool to room temperature, the solid was collected by filtration and washed with butyl acetate (1.2 mL), followed by drying under reduced pressure at 70-80° C. for 3 hours to obtain the Form II crystal (yield: 164 mg, percent yield: 58.8%, purity 96.04%).

Example 8 Synthesis of the Form II Crystal of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (400 mg) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide obtained according to the method of manufacturing described in International Publication No. WO2012/093708 pamphlet and International Publication No. WO2011/004610 pamphlet was added to cyclopentyl methyl ether (4.0 mL) and stirred for 3 hours by heating under reflux. After allowing the mixture to cool to room temperature, the solid was collected by filtration and washed with cyclopentyl methyl ether (1.2 mL), followed by drying under reduced pressure at 70-80° C. for 3 hours to obtain the Form II crystal (yield: 192 mg, percent yield: 68.8%, purity 95.68%).

Example 9 Synthesis of the Form II Crystal of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (400 mg) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide obtained according to the method of manufacturing described in International Publication No. WO2012/093708 pamphlet and International Publication No. WO2011/004610 pamphlet was added to propyl acetate (4.0 mL) and stirred for 3 hours by heating under reflux. After allowing the mixture to cool to room temperature, the solid was collected by filtration and washed with propyl acetate (1.2 mL), followed by drying under reduced pressure at 70-80° C. for 3 hours to obtain the Form II crystal (yield: 172 mg, percent yield: 61.5%, purity 96.77%).

Example 10 Synthesis of the Form II Crystal of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (400 mg) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide obtained according to the method of manufacturing described in International Publication No. WO2012/093708 pamphlet and International Publication No. WO2011/004610 pamphlet was added to methyl acetate (2.2 mL) and stirred for 16 hours by heating under reflux. After allowing the mixture to cool to room temperature, the solid was collected by filtration and washed with methyl acetate (0.94 mL), followed by drying under reduced pressure at 80° C. to obtain the Form II crystal (yield: 215.5 mg, percent yield: 68.6%, purity 98.06%).

Test Example 1 Blood Concentration Measurement Test

Respective solutions for administration (50 mg/10 mL/kg) of the Form I crystal and Form II crystal were produced. These solutions were administered orally to mice (Balb/cA) bred under feeding conditions at a volume of 10 mL/kg body weight using a sonde for oral administration. After the administration, the mice were returned to cages for mice to inspect their conditions. Water and feed were freely available for the mice in the cages. The mice were anesthetized with isoflurane 1, 2, 3, 4, 8 and 24 hours after the administration and 60 μL of blood was collected from their orbital sinuses using capillary blood collection tubes. The collected blood was cooled on ice and centrifuged to separate plasma.

AUCs_(0-24 hr)were calculated with a linear-log trapezoid method using Phoenix WinNonlin (v6.3.0), software from Pharsight, from the concentrations of Compound 1 in plasma samples measured with Multiple Reaction Monitoring method using LC-MS/MS.

Results are shown in FIG. 4 and Table 1. This test proved that the AUC_(0-24 hr) (area under the concentration in blood-time curve 0-24 hours after the administration) of the Form II crystal was approximately 3 times higher than that of the Form I crystal. Therefore, the Form II crystal according to the present invention is remarkably excellent in oral absorbability and useful as an oral pharmaceutical composition.

TABLE 1 Oral administration Form I Form II Parameter crystal crystal AUC_(0-24 hr) (μM · hr) 59.64 171.33

Test Example 2 Solid Stability Test (Accelerated Test)

The solid stability of the Form II crystal obtained in Example 1 after storage of 1, 3 and 6 months at 40° C.±2° C./75% RH±5% RH was measured under the following conditions.

-   Storage conditions: 40° C.±2° C./75% RH±5% RH -   Measurement points: 1, 3 and 6 month -   Storage amount: 6 g -   Storage container: doubled plastic bags, banding bands+plastic drums -   Preparation method of sample solution: 100 mg of the Form II crystal     is accurately measured, a mixed solution of acetonitrile-water (4:1)     is added to dissolve the crystal (sonicated to dissolve, if     difficult to dissolve), and the solution is diluted in measuring     cylinder to exact 200 mL total. 10 mL of this solution is accurately     measured, and a mixed solution of acetonitrile-water (1:1) is added     to exact 20 mL to be used as a sample solution.

HPLC was performed in the following conditions.

-   Column: general foundation Chemicals Evaluation and Research -   Institute, Japan L-column2 ODS -   Particle size: 3 μm, Inner diameter: 4.6 mm, Length: 15 cm -   Measurement wavelength: 220 nm -   Mobile phase: Mobile phase A: 10 mmol/L phosphate buffer (pH6.9), -   Mobile phase B: acetonitrile -   Flow rate: about 1.0 mL/min

The concentration gradient is controlled by changing the mixing ratio of mobile phases A and B as follows, when feeding the liquid.

TABLE 2 Time (min) after Mobile phase A Mobile phase B injection (%) (%)  0 to 25 80 20 25 to 45 80 → 65 20 → 35 45 to 55 65 35 55 to 70 65 → 10 35 → 90 70 to 80 10 90   80 to 80.1 10 → 80 90 → 20 80.1 to 95   80 20

The amounts of related substances in the sample solutions were measured and evaluated by HPLC, of which result is shown in Table 3.

The related substances are defined as substances detected other than 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide.

TABLE 3 1 month 3 months 6 months Related less than 0.1% less than 0.1% less than 0.1% substances in the Form II crystal

As apparent from this result, the Form II crystal of Compound 1 produces a low amount of related substances and shows excellent solid stability. 

1-11. (canceled)
 12. A method of treating a tumor, comprising administering, to a subject in need thereof, an effective amount of a Form II crystal of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide showing an X-ray powder diffraction spectrum having at least three characteristic diffraction peaks at angles (2θ±0.2°) selected from the group consisting of 7.7°, 8.0°, 11.1°, 12.5°, 12.9°, 15.2°, 15.8°, 17.2°, 19.0°, 22.5°, 26.1°, and 27.4°. 13-15. (canceled)
 16. The method according to claim 12, wherein the crystal shows an X-ray powder diffraction spectrum having at least five characteristic diffraction peaks at angles (2θ±0.2°) selected from the group consisting of 7.7°, 8.0°, 11.1°, 12.5°, 12.9°, 15.2°, 15.8°, 17.2°, 19.0°, 22.5°, 26.1°, and 27.4°.
 17. The method according to claim 12, wherein the crystal has an endothermic peak determined by differential scanning calorimetry of around 270° C.
 18. The method according to claim 12, wherein the crystal is in a pharmaceutical composition administered orally to the subject in need thereof.
 19. The method according to claim 12, wherein the tumor is a cancer selected from the group consisting of a head carcinoma, a neck carcinoma, a gastrointestinal carcinoma, a pancreatic cancer, a small intestine carcinoma, a large bowel cancer, a lung carcinoma, a breast carcinoma, an ovarian cancer, a uterine carcinoma, a kidney carcinoma, a bladder cancer, a prostate cancer, a urothelial carcinoma, a bone sarcoma, a soft tissue sarcoma, a blood cancer, a multiple myeloma, a skin cancer, and a mesothelioma. 